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The establishment of cell culture in hybrid type collagen and transplantation into intracerebral hemorrhage model rat brain

Research Project

Project/Area Number 10671300
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Cerebral neurosurgery
Research InstitutionKobe University

Principal Investigator

KONDOH Takeshi  Kobe Univ., School of Medicine, Dept. of Neurosurgery, Assistant Professor, 医学部, 助手 (50273769)

Co-Investigator(Kenkyū-buntansha) TAMAKI Norihiko  Dept. of Neurosurgery, Professor, Chairman, 医学部, 教授 (10030941)
Project Period (FY) 1998 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
KeywordsIntracerebral hemorrhage / Neuronal Transplantation / Blood-brain Barrier / GFP / HUVEC / Electroporation / コラーゲン培地 / 神経再生 / 細胞移植
Research Abstract

With a purpose of ex vivo gene transfer into injured brain such as intracerebral hemorrhage, following three sets of preliminary experiments were performed.
(A) gene transfer by electroporation was attempted in the normal brain. The reporter gene pEGFP-Cl(25g /5 μl) was injected in the striatum of young adult rats and various ranges of square electrical impulses were applied by using a pair of electrodes that were placed in the striatum. After five days, histological examination revealed that the impulses of high voltage caused extensive tissue damage whereas impulses of lower range (200-400 mJ) resulted in the transfection of more than 300 cells per brain, which were widely distributed in the subependymal region of the lateral ventricle and extended long processes into the striatum.
(B) Human umbilical vein endothelial cells (HUVECs) were transplanted in athymic mouse brain and neovascularization of grafled endothelial cells was studied. HUVECs were transfected by a reporter gene pEGFPE … More -N1 in vitro and grafted stereotactically in unilateral striatum of adult nude mice. Histological studies in four weeks revealed that grafted HUVECs newly formed microvessels in brain, which were migrated and fused with host vessels. Intravenous injection of Evans Blue prior to sacrificing animals resulted m no extravasation of dye, indicating that a blood-brain barrier was formed by the grafted HUVECs. Immunohistochemistry demonstrated that host astrocytes extended glial feet on the grafted endothelial cells and a part of the newly formed vessels were positive with glucose transporter-1. These results indicate that endothelial cells from an ectopic origin have the potential to form a blood-brain barrier after grafting in the central nervous system.
(C) Neuronal progenitor cells have been widly studied with the purpose of regeneration of injured central nervous system. For the grafting of these cells, not pure single cell suspension of stem cells but spheres composed with progenitor cells have been reported to demonstrate improved survival after grafting. In this study, rat neuronal stem cells were obtained from E-14 rat subventriclur zone followed by free floating culture in EGF-containing medium as previously reported by Weiss et al. After four passage in four weeks, single stem cells were left to grow without dissociation for two months by changing the medium weekly. The spheres became 500-800 um in diameter and then preserved in Hibernation Medium E (Gibco BRL) for a week at 4 ℃. Those sphere were labeled by PHK26 right before the grafting. Two different type of cerebral ischemia has been prepared in host adult rats. One is middle cerebral artery occulusion by phtochemical method and the other is endothelin injecton (ET-1 ; 0.05 ng/animal) into unilateral striatum. One week following transplantation of hibernated neuroprogenitor sphere demonstrated (A) no graft survival in the core of ischemic lesion in MCA occlusion model, (B) survival of small clusters in the border of ischemic core demonstrated by faint fluorscent marker, (C) dense clusters and migrating cells in the most brain in endothelin-injected striatum. Immunostaining using anti-MAP-2 and anti-GFAP serum demonstrated neuronal as well as glial cells to the grafts. Cryopreservation in DMSO-containing medium at-180 ℃ or preservation in hibernation medium longer than one week demonstrated flagile sphere which were unable to handle for grafting surgery. This study demonstrated that neuronal progenitor cell sphere are able to survive in the brain after preservation for one week. Less

Report

(4 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Kondoh T., et al.: "Long-term culture of neural stem cell spheres : A cytochemical and Ultrastractural study."Biomedical Research. 19. 209-216 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishizaki T., et al.: "Store Ca^<2+> depletion enhances NMDA responses in cultured human astrocytes."Biochemical and Biophysical Research Communications. 259. 661-664 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Wada T., et al.: "Ischemic "cross" tolerance in hypoxic-ischemia of immature rat brain."Brain Research. 847. 299-307 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Akiyama H., et al.: "Blood-Brain barrier formation of grafted human umbillical vein endothelial cells in athymic mouse brain."Brain Research. 858. 172-176 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kondoh T., et al.: "In vivo gene transfer into periventriclar region by electro - poration method."Med.Neuro.Chir(Tokyo). 40. 618-623 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishizaki T., et al.: "The anti-derentia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotine Ach receptors."Molecular Brain Research. 80. 53-62 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kondoh T, et al.: "Long-term culture of neural stem cell spheres : A cytochemical and ultrastractural study."Biomedical Research. 19. 209-216 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishizaki T, et al.: "Store Ca2+ depletion enhances NMDA responses in cultured human astrocytes."Biochemical and Biophysical Research Communications. 259. 661-664 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Wada, T, et al.: "Ischemic "cross" tolerance in hypoxic-Ischemia of immature rat brain."Brain Research. 847. 299-307 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Akiyama H, et al.: "Blood-brain barrier formation of grafted human umbilical vein endothelial cells in athymic mouse brain."Brain Research. 858. 172-176 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kondoh T, et al.: "In vivo gene trasnfer into periventriclar region by electroporation method."Neuro Med Chir (Tokyo). 40. 618-623 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishizaki T, et al.: "The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic Ach receptors."Molecular Brain Research. 80 (1). 53-62 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Akiyama H., et al.: "Blood-Brain barrier formation of grafted human umbilical vein endothelial cells in athymic mouse brain."Brain Research. 858. 172-176 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kondoh T., et al.: "In vivo gene transfer into periventriclar region by electro-poration method."Med.Neuro.Chir (Tokyo). 40. 618-623 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nishizaki T., et al.: "The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic Ach receptors."Molecular Brain Research. 80. 53-62 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nishizaki T,et al.: "Store Ca^<2+> dipletion euhamces NMDA nespeuses iu cultured human"Biochemical and Biophysical Researob Comwu*Icalus. 259. 661-664 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Wada T,et al.: "Ischewic "cross" tolerance in hypoxic-ischeuia"Braui Research. 847. 299-307 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Akiyama H,et al.: "BBB formation of grofted human wnbilical veiv evdothelial"Braui Research. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kondoh,T.et.al.: "Long-term culture of neural stem cell sphere:A cytochemical and ultrastractural studIes" Biomedical Research. 19. 209-216 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] 近藤 威、他: "In vivo electroporatoin法による正常ラット脳内遺伝子導入の試み" 神経組織の成長・再生・移植. 10. 7-8 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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